Intermediate Filaments (IFs) are one of three major classes of cytoskeleton found in every cell, and one of the more abundant cellular proteins. Mutations in IF proteins are responsible for at least 85 different human diseases, including many protein aggregation diseases. However, the unusual physical properties of these proteins have precluded determination of crystal structure. Because of this we have no in-depth mechanistic understanding of normal IF function, nor how mutations are pathogenic. In this proposal we will use biophysical approaches to define IF structure with a goal of elucidating IF function, and the degree to which IF structure is conserved across IF classes. This in turn will open the door to understanding pathogenesis among IF proteins, particularly those with protein aggregation phenotypes. We have assembled a team of several technologies: CW SDSL EPR, Pulsed Field SDSL EPR, Computational Modeling, High resolution TEM (Cryo- and STEM) and X Ray Crystallography. PUBLIC HEALTH RELEVANCE: Intermediate Filaments (IFs) are a major component of every vertebrate cell. IFs help cells resist mechanical and metabolic stresses, and have been implicated in more than 85 human diseases. However, we have no mechanistic understanding of IF biology because we lack structure for IFs. Moreover the same properties that make IFs stable, also contribute to their potential to cause proteins aggregation diseases. We will solve normal IF structure, so that we can understand the mechanisms of normal IF function, thus laying the groundwork for understanding how specific mutations are pathogenic.